Human respiratory syncytial virus (RSV), an enveloped RNA-containing virus of the paramyxovirus family, is the most important viral agent of pediatric respiratory tract disease. Its genome is a single negative strand of RNA of 15,222 nucleotides that encodes ten major mRNAs. The purpose of this project is to identify the functions of the viral proteins and to reconstruct events in the viral growth cycle under conditions where they can be more readily studied. We previously developed an intracellular transcription and replication system for RSV based on components expressed from transfected cDNAs. This involves transfecting tissue culture cells with plasmids which individually encode a helper-dependent minireplicon analog of negative-sense genomic or positive-sense antigenomic RNA as well as whatever mix of RSV proteins is desired. We previously used this approach to show that three proteins, the nucleocapsid N protein, phosphoprotein P and large polymerase subunit L protein, constitute the RSV replicase whereas, unexpectedly, the transcriptase requires in addition expression of the M2 mRNA. The M2 mRNA was shown to contain two overlapping translational open reading frames (ORFs), and the upstream one was shown to encode the processivity factor. Expression of the second ORF of the M2 mRNA inhibited RNA replication and transcription, providing functional evidence that this represents an eleventh RSV gene and is a novel negative regulatory factor. Also, the nonstructural protein NS1 appears to be a potent inhibitor of RSV RNA synthesis. We examined the widely accepted hypothesis that negative strand RNA viruses have a balance between transcription and RNA replication that is mediated by the availability of N protein, which is thought to bind to nascent RNA and shift RNA synthesis from transcription to RNA replication. Extensive analysis provided evidence for an alternative model in which transcription and RNA replication occur at a fixed ratio. We found that it is possible to reconstitute virion morphogenesis by coexpression of appropriate envelope components with the above-mentioned transcription and replication system. This showed that the matrix M, attachment glycoprotein G and fusion glycoprotein F protein are important for the formation of transmissible virus-like particles. The small hydrophobic SH protein, both ORFs of the M2 mRNA, and the nonstructural NS1 and NS2 proteins are completely dispensible for particle formation.